1. Field of the Invention
The present invention relates to an immunoassay system which utilizes hybridomally produced high affinity IgM antibodies.
2. Description of the Prior Art
The use of immunoassay for the detection of minute amounts of substances in physiological fluids is very well known in the art. The assay basically depends on the binding interaction between an antigen and an antibody therefor. In the great majority of systems described in the prior art, the antibody is usually immunoglobulin G (IgG). Among these, the polyclonal IgG's have until recently been routinely utilized (see for example T. Chard, "An Introduction to Radioimmunoassay and Related Techniques", North-Holland Publishing Company, 1978).
The advent of hybridomal techniques has brought about the possibility of producing homogeneous populations of highly specific antibodies against a variety of antigens. Thus, Koprowski et al U.S. Pat. No. 4,172,124 describe antibodies demonstrating specificities for malignant tumors produced by somatic cell hybrids between myeloma cells and spleen or lymph cells. Koprowski et al U.S. Pat. No. 4,196,265 describe continuous cell lines of genetically stable fused cell hybrids capable of producing large amounts of IgG antibodies against specific viruses, such as influenza virus, rabies, mumps, SV40, and the like. Zurawski et al, Federation Proceedings 39:4922 (1980), disclose hybridomas producing monoclonal IgG antibodies against tetanus toxin. Frankel and Gerhard, Molecular Immunology, 16:101-106 (1979) also disclose monoclonal antiviral hybridoma IgG antibodies described as "true bivalent antibodies", Frankel and Gerhard, at 105. The Frankel and Gerhard paper describes what are probably the prior art antibodies having the highest affinities to date. The monoclonal antibodies of Frankel and Gerhard have K values (binding constants determined from Scatchard plots) in the ranges of from less than 10.sup.5 M.sup.-1 to about 10.sup.10 M.sup.-1. The aforementioned Zurawski et al paper describes monoclonal antibodies having K association constants in the range of 3.5-5.4.times.10.sup.8 M.sup.-1.
The use of IgM antibodies has received relatively less attention in the immunoassay art. IgM antibodies consist of 5 units each the approximate size of IgG joined by a so-called J (joining) chain. The IgM molecule has a valency of 10, although quantitative antigen binding measurements often detect only 5. IgM's are extremely sensitive to reducing agents, they self-aggregate readily and precipitate out of solution, and have been found to bind in a non-specific manner. These reasons, among others, have heretofore prevented their extensive use. Because of the polyvalency of IgM antibodies, various suggestions have been made in the art concerning the theoretical binding affinity of these antibodies towards antigens. For example Metzger, has suggested that it would be "intuitively obvious" that an antibody with multiple binding sites would bind more firmly than a univalent or bivalent antibody to a multivalent antigen. Metzger however qualifies this statement recognizing that the energetics of binding would be highly dependent on the number and topology of antigenic determinants, the distribution of combining sites in space and the balance between the free energy of binding released by combining site/determinant interactions and high free energy required to maximize the number of multiple flexible conformations existing in such large molecules. These qualifications are even more stringent when it comes to cross linking of translationally independent determinants such as in agglutination. Mathematical models have been developed describing the theoretical binding of multivalent IgM antibodies to multideterminant antigen particles (Crothers and Metzger, Immunochemistry, Volume 9, 341-357 (1972)). It has even been suggested (Ehrlich, Journal of Theoretical Biology, 81:123-127 (1979)) that multivalency might theoretically increase specificity. All of these expectations of the art, as represented by the intuitive feelings and purely theoretical mathematical models, have been at odds with the experimental facts except in certain specific instances, infra. Although IgM antibodies are multivalent, the binding affinities observed for them have, prior to this invention, always been substantially less than the binding affinities for IgG antibodies. It is for this reason that IgM's have not routinely been utilized for immunoassays, other than in certain specific circumstances, infra. The art has explained the observed poor binding affinity of IgM antibodies, as being due to the structural features of the molecules, and the probability that combining sites of IgM molecules have lower avidity for antigens than do those of IgG's. Cunningham, A. ("Understanding Immunology", Academic Press Inc., 1978, at 33), in suggesting why quantitative antigen binding measurements with larger antigens on IgM antibodies detect only 5 and not 10 binding sites, discusses the possibility that for reasons of steric restriction, all the sites cannot attach to the same antigenic structure at the same time.
Faced with the lesser utility of IgM antibodies in conventional precipitation or immunoassay techniques, the prior art nevertheless details some special circumstances under which this class of antibodies can, and has been used. Soothill et al U.S. Pat. No. 4,210,622 describe the use of heterologous low affinity IgM obtained from myeloma serum for the selective determination of immune complexes. This technique takes advantage of the fact that low affinity IgM's will selectively bind antibody-antigen complexes even in the presence of individual components of the complex. Soothill et al U.S. Pat. No. 4,141,965 describe a method wherein low affinity heterologous IgM antibodies are used to agglutinate antigen-coated latex particles. When physiological fluid containing antigen is added to the agglutinate mixture, an inhibition-of-aggluination occurs. Gopalakrishnan and Karush, Journal of Immunology, 113: 769-778 (1974), disclose the use of IgM antibodies of restricted heterogeneity induced by a lactoside determinant, for the binding of conjugates of the lactoside determinant with bacteriophage .phi.X174. These authors report that the constant for the binding of the antibody to the conjugate (.about.10.sup.9 M.sup.-1) is greater by a factor of 10,000 than that for the lactoside itself (.about.10.sup.5 M.sup. -1). Blank et al, Journal of Immunology, 108:665-673 (1972)) disclose affinity constants for grouper IgM's in the order of 10.sup.4 M.sup.-1. A specific instance of an IgM antibody, rheumatoid factor (RF) has also been utilized for immunoassays in the past. RF's are IgM antibodies directed towards antigenic determinants present on autologous immunoglobulin Lurhuma et al, Clinical and Experimental Immunology, 25:212-226 (1976), describe the use of RF as an agglutinant for IgG coated particles, and an inhibition of aglutination effect which occurs in the presence of antibody-antigen complexes. Masson et al U.S. Pat. No. 4,062,935 describe a similar system to that of Lurhuma.
Monoclonal RF to human IgG has been used by Eisenberg, Immunochemistry, 13:355-359 (1976) to demonstrate binding to aggregated IgG. The binding of monoclonal RF to aggregated IgG is increased by a factor of greater than 10.sup.6 (as determined by radioimmunoassay) over the binding of RF to nonaggregated, monomeric, IgG.
In sum, the use of IgM antibodies, whether polyclonal or monoclonal has been restricted to assaying for antibody-antigen complexes, aggregated IgG's, or, by an inhibition of agglutination technique, to antigen-coated latex particles. Because of the poor binding affinity of IgM's as well as their self-aggregating tendency, non-specific interactions and sensitivity to reducing agents, it has generally been assumed in the art that IgM antibodies were not useful for highly sensitive immunoassay techniques.